Project Summary/Abstract The accurate segregation of chromosomes ensures the faithful inheritance of genetic information. Defects in chromosome segregation can cause an imbalance in chromosome number, or aneuploidy, which is the leading cause of over 90% of human cancers and contributes to spontaneous abortion and birth defects (e.g. Down syndrome). The locus that ensures that chromosomes are equally segregated during cell division is the centromere. Centromeres are comprised of repetitive ?-satellite that span several megabases on each chromosome. The repetitive nature of these regions has posed an enormous challenge to standard short-read sequencing and assembly methods, and as a result, all centromeres remain unassembled in the human genome. The lack of centromere sequence assemblies has greatly hindered our understanding of the role these sequences play in essential cell biological processes required to maintain genome integrity. This proposal aims to address this gap in knowledge by generating linear sequence assemblies of each human centromere using a combination of long-read sequencing technologies and novel computational assembly tools. The proposed work will also reconstruct the evolutionary history of centromeres by elucidating the genetic variation of centromeres in humans and apes. In addition, this work will uncover how genetic variation at centromeres impacts the transcriptional landscape by sequencing and annotating full-length centromeric transcripts in the human genome. Finally, this proposal will determine if genetic variation at centromeres impacts the ability of chromosomes to be accurately segregated during cell division using cell-based assays. Taken together, this research will uncover the linear organization of human centromeric regions and elucidate how genetic variation in these regions impacts the accuracy of chromosome segregation during cell division.